Meso-四(3,5-二溴-4-羟基苯基)卟啉褪色光度法测定蛋白质的研究与应用

Study and Application of Determination of Protein with Meso-Tetra-(3,5-Dibromo-4-Hydroxyphenyl) Porphyrin Fading Spectrophotometry

采用吸光光度法研究了meso-四（3，5-二溴-4-羟基苯基）卟啉[T（DBHP）P]作为新型光谱探针与蛋白质相互作用的光谱行为。发现在pH4．17Britton-Robinson缓冲液中，吐温-80（Tween-80）微乳液介质可以显著增强了体系的灵敏度。在最佳实验条件下，考察了T（DBHP）P-蛋白质体系的吸收光谱特性，于425nm处蛋白质的浓度在一定范围内与吸光度A呈良好的线性关系。分别用于测定牛血清白蛋白（BSA）和卵血清白蛋白（Ova），线性范围分别为0．50～6．00μg·mL^-1和0．05-0．60μg·mL^-1，检出限分别为0．11和0．039μg·mL^-1。实验结果表明，该方法具有较高的灵敏度、选择性和稳定性，成功地用于牛奶样品中蛋白质的测定，回收率为99．56％～100．2％，相对标准偏差低于2．2％。从而建立了一种测定蛋白质的灵敏方法并用于食品分析领域中。另外考察了离子强度及温度对体系的影响。

In the present paper, the binding characteristics and spectral behavior of interaction of meso-tetra-（3, 5-dibromo-4- hydroxyphenyl） porphyrin ET（DBHP）P] as a new-style probe with protein were studied by the techniques of spectrophotome- try. The experiment showed that Tween-80 microemulsion was efficiently used to enhance the sensibility and stability of the system at pH 4. 17（Britton-Robinson buffer solution）. Under optimum conditions, the characteristics of absorption spectral of meso-tetra-（3,5-dibromo-4-hydroxyphenyl） porphyrin-protein were investigated, the maximum absorption was located at 425 urn, and the reducing value of absorbance A was in proportion to the concentration of proteins in the range 0. 50-6.00/μg · mL^-1 for bovine serum albumin, 0. 05-0. 60 μg · mL^-1 for ovalbumin, and the limits of detection were 0. 106 μg · mL^-1 for bovine serum albumin and 0. 039 μg · mL^-1 for ovalbumin. The experiments indicated that the proposed method featwed high sensitivity and good selectivity and stability, and was simple and relatively free from interference of coexistent substances. It has been applied to the determination of protein in milk samples with satisfactory. The recovery for the investigated protein from milk was 99. 56%- 100. 2% and the relative standard deviations were less than 2. 2%. The sensitive method for the quantitative determination of proteins was proposed and may be applicable to the determination of ultra amounts of protein in food analysis. The effect of ionic strength on the system was investigated, and the result indicated that the binding force between meso-tetra-（3, 5-dibromo-4- hydroxyphenyl） porphyrin and protein was judged as electrostatic force. The influence of protein denaturation was also studied, under higlaer temperature the structure of protein was destroyed, and thermodynamic movement of the molecular of protein was intensified as the heating time extended.